Erythromycins are members of the polyketide class of natural products that also includes many important antibiotic, antifungal, anticancer, and immunosuppressive compounds (Staunton and Wilkinson Chem. Rev. (1997) 97, 2611-2629). This class of compounds has attracted considerable interest because of the wide range of biological activities these compounds possess, and their potential to provide therapeutic drugs across a wide range of disease states.
More recently the discovery that these compounds are synthesised by the repeated stepwise condensation of acyl-thioesters in a manner analogous to that of fatty acid biosynthesis to generate chains of varying length and substitution has attracted research towards uncovering the enzymic mechanisms by which these molecules are put together. FIG. 1 shows how erythromycin is assembled from these basic ketide units. The structural diversity found among natural polyketides arises in part from the selection of (usually) acetate (malonyl-CoA) or propionate (methylmalonyl-CoA) as “starter” or “extender” units (although one of a variety of other types of unit may occasionally be selected). Further structural diversity is given by the differing degree of processing of the β-keto group formed after each condensation. Examples of processing steps include reduction to β-hydroxyacyl-, reduction followed by dehydration to 2-enoyl-, and complete reduction to the saturated acyl-thioester. The stereochemical outcome of these processing steps is also specified for each cycle of chain extension. Methylation at the α-carbon or at the oxygen substituent is also sometimes observed. These processes are catalysed and controlled by a polyketide synthase. Other enzymes add further structural diversity after the completed polyketide chain is released from the PKS, for example, methylation, oxidation or glycosylation. A number of types of polyketide synthase have been identified, while these synthases catalyse broadly similar reactions they act in very different ways and have significantly different quaternary protein structures.
A wide range of polyketides has been identified from natural sources, and it is believed that only a small fraction of the potential diversity from the natural pool has been sampled to date. There is a clear interest in the discovery of alternative routes to find or generate polyketides of novel structure. The discovery that the biosynthesis of Type I polyketides occurs in an ordered fashion (Cortés et al., Nature (1990) 348, 176-178, Donadio et al., Science (1991) 252, 675-679, Donadio et al., Gene (1992) 111, 51-60, Donadio et al., PNAS (1992) 90, 7119-7123, WO93/13663) and the subsequent experiments that showed that portions of the genes encoding these large multifunctional proteins could be swapped between proteins to produce a ‘hybrid PKS’ has greatly expanded the potential repertoire of molecules that can be produced by biological routes (U.S. Pat. No. 6,271,255; WO 98/01546). For example, it is possible to replace each of the methylmalonyl-CoA specific acyltransferases of the erythromycin cluster with an AT specific for malonyl-CoA to produce a hybrid PKS-containing strain that can biosynthesise a macrolide polyketide core lacking a methyl group at the expected position (WO 98/01546, Petkovic, H et al, 2003, J Antibiot (Tokyo). 56(6):543-51). Similarly, it is possible to substitute or alter the reductive loops to leave an altered oxidation state at positions around the ring (WO 98/01546, WO 98/01571, and WO 00/01827). Such techniques involving the genetic engineering of a Type I PKS are applicable to all polyketides synthesised by such Type I enzymes.
In many cases to gain a commercially useful compound the polyketide is used as a template for semi-synthetic derivatisation. In addition to altering/modifying the activity of the molecule such derivatisation might confer a range of properties on the molecule that affects its pharmacological properties, for example but without limitation, altered bioavailability or stability.
One of the important aspects of genetic manipulation of the systems that biosynthesise such molecules is the ability to provide a chemical ‘handle’ for the addition of such derivative groups, for example replacement of a methylene group by a hydroxyl group by substituting the components of the reductive loop. The ability to feed such a modified polyketide into a combinatorial chemistry program greatly increases the number of altered molecules that are generated and that can be screened for the optimal activity or indeed for entirely different properties/activities. In addition however, the underivatised manipulated molecule may also have desirable properties over the original molecule.
Erythromycin is one of the key members of a class of polyketide molecules containing a 14-membered macrolide ring. Other members of this class include, without limitation, oleandomycin, megalomycin, narbomycin and pikromycin (Shiomi, K & Omura S, 2002, Macrolide Antibiotics: Chemistry, Biology and Practice, Ed. Omura, S, Academic Press, 2nd Edition, pp 1-56). Different members of the class of 14-membered macrolides are able to display a range of oxidation states at positions around the ring and a range of substituent groups (for example but without limitation, H—, methyl-, methoxy- or ethyl-) around the ring. 14-membered macrolides can be modified by addition of other groups such as glycosyl groups or other modifications (e.g. methylation, acetylation etc). Other modifications have been introduced chemically (Sunazuka, T et al, 2002, Macrolide Antibiotics: Chemistry, Biology and Practice, Ed. Omura, S, Academic Press, 2nd Edition, pp 99-180).
Many of the naturally available chemical handles on the erythromycin molecule have already been used to produce altered erythromycins. For example the 6-hydroxy group has been methylated to produce the commercially available antibiotic clarithromycin. The same position has been modified using a range of other groups to produce compounds such as ABT-773 via the C6-O-allyl intermediate (WO 98/09978; Ma et al., J. Med Chem (2001) 44, 4137-4156). Additionally, it has been reported that the 11- and 12-hydroxy groups have been derivatised (Ku et al., J. Antibiotics (1999) 52, 908-912). The secondary and tertiary hydroxyl groups at C11 and C12 respectively have been used to produce the C11, C12-cyclic carbamate structures seen, for example in telithromycin (Bryskier A., (2000) Clin Microbiol Infect. 6(12): 661-9). The 9-keto group is used to produce the Azithromycin derivatives and has been used to prepare C-9 amino ketolides (Ballow C H & Amsden G W, 1992, Ann Pharmacother.; 26(10):1253-61; Retsema J and Wenchi F; 2001, International Journal of Antimicrobial Agents, 18, pp S3-S10). Removal of the deoxyhexose moiety at C3 and oxidation of the resulting secondary hydroxy group to a keto group results in the ketolide series. Additionally, the available chemical handles on the sugar moieties have also been modified. Kaneko and co-workers (Kaneko et al., Exp. Opin. Ther. Patents (2000) 10, 1-23) have reviewed some of the recent developments in the area of macrolide antibiotics and describe many other similar modifications around the molecule although this is unlikely to be an exhaustive list of modifications that are possible.
The ability to further increase the number of chemical handles around the molecule would result in novel series of erythromycin compounds by utilization of such handles for semi-synthetic derivatisation. Indeed, many of the chemical methods currently utilising the existing handles may also be used to derivatise new chemical handles, if they could be built into a molecule. Additionally, if it is possible to build into a molecule a chemical handle of a type that does not already exist, then it may be possible to use a different array of chemistries. For example, it will be understood that it would be beneficial to introduce a primary hydroxyl group if a molecule only contained secondary or tertiary hydroxyl groups as this would allow a chemist to specifically derivatise the primary hydroxyl group using chemical methods specific for primary alcohol groups.
A series of compounds of particular interest are those containing alterations in the starter unit area of the molecule (U.S. Pat. No. 6,271,255; WO 98/01546). In the natural erythromycin molecule this starter unit comprises a C13-ethyl group. This is generally considered an unreactive area of the molecule and hence is unavailable for conventional chemical modification, especially in the context of such a molecule that contains many other, more labile, chemical groups. Consequently, a number of novel techniques have been devised to produce alternative polyketide 14-membered macrolide molecules, and particularly erythromycins possessing altered starter units, for example: replacement of the loading module (U.S. Pat. Nos. 6,271,255, 6,437,151, WO 98/01571, Marsden et al., Science (1998) 279, 199-202; Pacey et al., J. Antibiotics (1998) 51, 1029-1034) or N-acetyl cysteamine ester feeding (Jacobsen et al., Science (1997) 277, 367-369). These techniques may require the feeding of precursors to strains that are modified in some way to accept the novel substrates. In some cases the supply of natural substrates is inhibited to allow the acceptance of the novel substrate. However, these techniques are limited as the natural degradative pathways in the cell (e.g. β-oxidation, lipase) compete with the secondary metabolic pathways to reduce the effective amounts of the precursor compounds available and hence the final yield of desired compound in the cell (Frykman et al., Biotechnol. Bioeng. (2001) 76, 303-310). A further disadvantage of these methodologies is that competition of the fed starter with the natural endogenous starter substrates can result in a mixture of products (Pacey et al., J. Antibiotics (1998) 51, 1029-1034).
Other methods that could be used to produce 14-membered macrolides with hydroxylated starters include increasing the numbers of extension cycles that a Type I PKS responsible for the formation of a 14-membered macrolide performs. This can lead to the formation of an octaketide molecule which may cyclise to form a 14-membered macrolide with an extended exocyclic moiety. Methods for producing such molecules are detailed in for example (WO 93/13663, U.S. Pat. No. 6,271,255; WO 98/01546; WO 99/36546; Rowe et al., Chem. Biol. (2001) 8, 475-485). However, Rowe et al., (Chem. Biol. (2001) 8, 475-485), indicate that the introduced extension cycle is sometimes skipped, and that a mixture of molecules including both 14- and 16-membered molecules is produced due to different possible cyclisation patterns that are possible for a linear polyketide molecule catalysed by the polyketide chain termination/cyclisation domain (thioesterase).
Therefore, there is a need to develop alternative methods of generating altered starter units or for activating the starter unit-derived portions of 14-membered macrolides such as erythromycin to provide molecules or templates for molecules with altered antibacterial or other pharmaceutical activity.
One alternative method to activate the starter unit of a polyketide would be to use an enzyme that specifically hydroxylates the starter unit of a polyketide. One potential candidate for this type of enzyme is a cytochrome P450. These enzymes possess a strong oxidizing potential and it is known that some such enzymes are able to act in the later stages of polyketide biosynthesis. Wide ranges of cytochrome P450 hydroxylases have been identified, including many that act specifically in polyketide biosynthetic pathways. It is also known that cytochromes P450 play a significant role in the degradation of drug-like substances in mammalian systems and they act similarly in bacterial cells through catabolic/xenobiotic pathways. They are also known to act on agrochemicals and environmental pollutants. Key to this approach is to develop a system that is efficient and that can act relatively specifically to the starter unit. Other oxidative enzymes such as flavin dependent monooxygenases and non-heme iron dependent dioxygenases exist.
Cytochromes P450 show a high degree of stereo- and regiospecificity, which can have wide industrial application including the production of statins and corticosteroids. Cytochromes P450 have been used for the preparation of valuable drug metabolites and structural studies of human cytochromes P450 are aiding the design of drugs.
A number of cytochromes P450 that act on 14-membered macrolides have been identified and cloned. These have been shown to act on glycosylated or non-glycosylated macrolide substrates depending on the specificity of the enzyme. For example:                EryF—which acts at the C6 position of the erythromycin aglycone 6-dEB (6-deoxy erythronolide B, Andersen, J. F. and C. R. Hutchinson (1992). Journal of Bacteriology 174(3): 725-735).        EryK—which acts at the C12 position of erythromycin D, i.e. after glycosylation at the C3 and C5 hydroxy groups (Stassi, D., S. Donadio, et al. (1993). Journal of Bacteriology 175(1): 182-189).        OleP—which acts to introduce an epoxide group at the C8, C8a position during biosynthesis of oleandomycin (Shah, S., Q. Xue, et al. (2000). Journal of Antibiotics 53(5): 502-508).        PikC hydroxylase—which has a dual specificity, acting at C12 of the 14-membered narbonolide structure and at C10 and C12 of the 12-membered 10-deoxymethynolide structure (Xue, Y. Q., D. Wilson, et al. (1998). Chemistry & Biology 5(11): 661-667). Note that this oxidation results in the hydroxylation of the starter unit region of the 12-membered methynolide but this does not appear to occur on the 14-membered narbonolide        
Cytochromes P450 acting at various positions on 16-membered macrolide rings such as tylosin are well documented; again said oxidation can occur before or after glycosylation. However, to date although a small number of cytochromes P450 acting on the starter unit region of glycosylated 14-membered macrolides such as erythromycin have been identified, they have not been well described. Additionally, to date no cytochromes P450 which act to hydroxylate the starter unit region of unglycosylated 14-membered macrolides such as erythronolides have been described. Examples of 14-membered macrolide natural products do exist in which the starter unit region appears to be hydroxylated; e.g. CP-63693 (U.S. Pat. No. 4,543,334) and lankamycin. However, it is not known whether such oxidations occur before or after glycosylation, indeed in the lankamycin case this hydroxyl could in principle be polyketide derived (as opposed to introduced after polyketide formation). Recently the entire biosynthetic cluster for lankamycin has been published, revealing the presence of two encoded cytochromes P450 although it is not known at what position each of these act (C8 or C15) and analysis of the PKS shows that 2-methylbutyrate is a likely starter unit (Mochizuki et al., Mol. Microbiol. (2003) 48, 1501-1510).
Recently, the structural determination of the 50S subunit of the ribosome has shown that the nature of their sugar attached has a strong influence on the binding of macrolide antibiotics to the ribosome (Harms et al., Cell (2001) 107, 679-688; Schlunzen et al., Nature (2001) 413, 814-821; Hansen et al., Mol. Cell (2002) 10, 117-128). Therefore, there is a need to develop methods of producing 14-membered macrolides that possess altered glycosylation patterns. It is thought such information on the binding of the macrolides to the ribosome will greatly aid the rational design of macrolide antibiotics that might overcome the typically encountered resistance mechanisms such as methylation or modification of residues/nucleotides within the ribosome. Such studies also demonstrate a significant contribution to binding from the starter unit derived portion of the molecule, hence it is an advantage to be able to alter the nature of the starter unit derived portion and/or the glycosyl groups attached.
The present invention provides a method to combine functional changes at the starter unit region (e.g. hydroxylation at the 14- or 15-position of 14-membered macrolides) with an ability to transfer a range of different glycosyl groups. This method enables the production and isolation of a diverse range of novel macrolide molecules, which may be then optionally be tested for optimal functionality.
In one aspect the present invention provides a method for the generation of hydroxylated 14-membered macrolide compounds, said method comprising producing a 14-membered aglycone template and feeding said 14-membered aglycone template to a strain or range of strains to produce a range of novel compounds that differ in the hydroxylation state of the starter unit region. In one embodiment, the aglycone template is fed to a single strain. In an alternative embodiment, the aglycone template is fed to a range of strains.
In a further aspect, the present invention additionally provides a method for the generation of 14-membered macrolide compounds, said method comprising feeding a hydroxylated aglycone precursor generated as described above, to a strain or range of strains to produce a glycosylated compound or range of compounds containing a range of sugar moieties.
Biotransformation of erythronolide B to produce a compound hydroxylated at the 15-position has been shown previously (Spagnoli et al., J. Antibiotics (1983) 36, 365-375; U.S. Pat. No. 4,429,115). This system used a blocked mutant of S. antibioticus (which normally produces the 14-membered macrolide oleandomycin) as the biotransforming organism. A mixture of 4 compounds were produced, 3-O-oleandrosyl-5-O-desosaminyl-15-hydroxyerythronolide B, 3-O-oleandrosyl-5-O-desosaminyl-erythronolide B, 3-O-oleandrosyl-5-O-desosaminyl-(8S) 8-hydroxyerythronolide B and 3-O-oleandrosyl-5-O-desosaminyl-(8R) 8, 19-epoxyerythronolide B (U.S. Pat. No. 4,429,115). In this case it was determined that the hydroxylation occurred after glycosylation (Spagnoli et al., J. Antibiotics (1983) 36, 365-375). It is noteworthy that hydroxylation was observed at both the C8 and C15 positions of the macrolide ring.
Biotransformation of the 14-membered macrolide 11-acetyllankolide (a 14-membered macrolide aglycone) using a blocked mutant of the erythromycin producing organism Streptomyces erythreus (since reclassified as Saccharopolyspora erythraea) resulted in production of 15-deoxy-15-oxo 11-acetyllankolide (Goldstein et al., J. Antibiotics (1978) 31, 63-69). In this case oxidation of the starter unit region does occur on an unglycosylated 14-membered aglycone, although the starter unit region is already functionalised with a hydroxy group present at the 15-position; oxidation converts the hydroxy group to a keto group.
14-hydroxyclarithromycin is observed as a metabolite of clarithromycin produced by the action of one of the major cytochromes P450 in the liver. Obviously, in vivo, hydroxylation of clarithromycin must occur after glycosylation. Additionally, methods that have been described for the synthesis of 14-hydroxyclarithromycin and related compounds have involved hydroxylation of the active glycosylated compound (EP 0 222 186, U.S. Pat. No. 4,672,056, Adachi et al., J. Antibiotics (1988) 16, 966-975). In these cases a range of compounds are produced and the route to the compound (feeding erythromycin A to human volunteers and extracting the metabolites from their urine) is unlikely to represent a large-scale production route. As the inventors of EP 0 222 186 acknowledge, this does not occur, for example, using homogenates of rat liver, further reducing the likelihood that it will result in a cost effective route. A route to produce 14-hydroxyclarithromycin from clarithromycin by bioconversion using the fungus Mucor circinelloides has been shown (Sasaki et al., J. Antibiotics (1988) 41, 908-915).
In the two previous examples of 14- or 15-hydroxylation of 14-membered macrolides, glycosylation occurred prior to hydroxylation, therefore at no point was the hydroxylated aglycone precursor formed. Additionally, the sequence of events results in a limited ability for a person skilled in the art to select the sugars that are attached in each case. In contrast, the present invention provides for the synthesis of a hydroxylated aglycone template that can then be fed to a wide range of natural or recombinant strains, resulting in the addition of a range of sugars and the generation of a wide range of compounds. The present invention is the first example, to our knowledge, of the generation of erythromycin analogues that possess hydroxylation on the starter unit region via hydroxylation of the aglycone template, which is later glycosylated. Previous studies had assumed that glycosylation occurred first and therefore have not conceived of the present methodology.
The present method has advantages over methods that produce hydroxylated compounds by engineering the polyketide synthase as it does not require the addition of precursors that might be degraded and since the 14-membered macrolide is already formed before hydroxylation it reduces the chance that cyclisation can occur to give a 16-membered ring.
The present invention additionally teaches that once a cytochrome P450 has been identified that is capable of hydroxylating the starter unit region of a 14-membered aglycone it would be possible to clone the gene encoding the cytochrome P450 and express this gene in an alternative host. Thus by expressing such a gene in a host capable of producing the 14-membered aglycone it is possible to produce a strain able to produce the appropriately hydroxylated 14-membered aglycone directly. Similarly it would be possible to engineer a strain that is able to perform both the hydroxylation of the starter unit region and the subsequent glycosylation of the hydroxylated 14-membered aglycone
The present invention provides methods for transforming the starter unit region of the aglycone of a 14-membered macrolide biosynthetic pathway by biotransformation and, optionally, subsequent processing of the molecule so produced into a glycosylated form by the addition of one or more sugar moieties; and molecules produced by the methods of the present invention.
The 14-membered macrolide analogues of the present invention also possess further utility as intermediates for the synthesis and semi-synthesis of antibacterial C14- or C15-hydroxyerythromycin or other 14-membered macrolide derivatives.